We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

所有的动物实验是按照卫生指南实验动物的护理和使用国家研究院​​（NIH出版物号80-23 2012年修订），并研究批准了机构动物护理和使用研究委员会石溪布鲁克大学（＃1618和＃1792）。 1. vHipp-NACC突触共培养<ol><li>牺牲小鼠（出生后第0天- 3，从野生型（WT）或α7烟碱受体的转基因小鼠品系）用CO 2。斩首每个小狗和保存的尾部在1.5毫升离心管，用于事后的基因分型。执行在无菌罩下面的步骤。 </li><li>除去用剪刀和镊子的颅骨顶部以暴露整个大脑。开始于时刻所在的大脑皮层部分彼此尾端，得到冠状部，其包含的后皮质包括vHipp 5,21在解剖显微镜下。 </li><li>解剖v含有CA1〜下脚区域出根据显影小鼠脑图谱22（ 图1A）希普组织条。转移到含有冷培养基（4℃）35毫米培养皿，切成小薄片（约100微米×100微米microslices）。 </li><li>预孵育12毫米聚-D-赖氨酸/层粘连蛋白包被的玻璃内的24孔组织培养板用培养基（〜500微升）的盖玻片在培养箱中在至少30分钟，在37℃。电镀microslices前取出介质。 </li><li>板用火抛光的巴斯德吸移管在用媒体最小量盖玻片的中心（约50微升，含有〜20件microslices的），以促进附着外植体。板vHipp microslices从一个单一的动物起源（无论是+ / +或 - / - 基因型转基因α7线）上的每个盖玻片。 </li><li>后定居植到盖玻片，轻轻日增加额外的媒体（约100微升）Ë壁上，使得介质的水平高到足以完全覆盖在周边的外植体，而不是那些在盖玻片的中心。 </li><li>的O / N培养在37℃后，5％CO 2培养箱，分散NACC神经元从胚胎天18到产后第1天WT小鼠，并加入到植。 <ol><li>根据显影小鼠脑图谱20（ 图1B）解剖出来NACC组织。 </li><li>切成小块（约500微米×500微米microslices），并转移到15毫升管。 </li><li>治疗与0.25％胰蛋白酶（2ml）中进行15分钟，在37℃。洗涤组织块接着在冷的培养基一个洗涤三次用冷洗涤介质（5毫升，4℃）（5毫升，4℃）。允许悬挂休息5分钟，每个洗涤步骤，然后小心地倒出上清液。 </li><li>游离细胞轻轻研磨在2毫升培养基用轻火抛光的巴斯德吸管。 </li><li>反FER细胞悬液至另一个15ml试管并离心以2,000×rpm离心5分钟。去除上清和悬浮细胞培养液中，每6幼仔1毫升。在媒体几次轻轻吹打用火抛光的巴斯德吸管分散细胞。 </li><li>添加0.25毫升分散NACC细胞对每个盖玻片与vHipp植。 </li><li>保持培养物在加湿37℃，5％CO 2培养箱。广场上蘸消毒纱布垫培养板机械稳定和保持湿度，促进突触的形成。 </li></ol></li></ol> 2.免疫细胞化学<ol><li>固定培养物（5 - 7日在体外 ）在4％低聚甲醛/ 4％蔗糖/ PBS（〜每盖玻片500微升，20分钟，室温）。 </li><li>透培养用0.25％的Triton X-100 / PBS（〜每盖玻片500微升，5分钟，室温）。 </li><li>块培养物用PBS中的10％正常驴血清（〜每盖玻片500微升，30分钟，室温），以antibod之前ÿ染色。孵育一抗O / N在4℃。使用下列一抗：抗nAChRs的（α4 -ECD 1：500;抗α5亚基，1：500），抗囊泡膜谷氨酸转运蛋白1（1：250），抗GAD 65（1：100）。 </li><li>洗一次抗体用PBS（3次/〜500每盖玻片微升，5分钟），然后孵育培养物中结合Alexa 488二抗（〜每盖玻片500微升，1：500）或Alexa 594（〜500微升每盖玻片，1：500），1小时，在37℃。 </li><li>固定之前用于标记表面α7* nAChRs的45分钟，在37℃：孵育培养物中αBgTx缀合的Alexa 594（1000在培养基〜每盖玻片500微升，1）。 </li><li>安装和密封盖玻片。 </li><li>用装有计划复消色差透镜的目标（20X用0.8的NA或63X油1.4 NA）为荧光显微镜和CCD照相机捕捉图像。 </li></ol> 3.基于FM1 - 43水疱融合<ol><li>保持文化（5 - 7日在体外 ）在成像室，安装室内的旋转盘共聚焦显微镜。连续灌注（1毫升/分钟）与哈佛商学院的鸡尾酒在室温。 </li><li>停止灌注和负载的文化与10μMFM1 - 43在56毫米的K +学联（〜500微升），90秒（染色），15分钟与钙含ADVASEP-7（0.1毫米）免费洗HBS外部客场染料以清除膜结合FM1-43。 </li><li>刺激的培养物用56毫K + ACSF（〜500微升），而不FM1-43 120秒（脱色），用同样的条件下重新加载培养用FM1-43，并与钙洗一遍2+含有游离的HBS ADVASEP-7为15分钟。 </li><li>获得FM1-43荧光图像与旋转盘共聚焦显微镜配备一个计划复消色差透镜物镜（60X水与1.4的NA，激发488纳米，发射530 nm），并将捕获的图像用CCD照相机。 </li><li>收集基线FM1 - 43荧光图像（每2秒1分钟）作为预尼古丁控制;应用尼古丁（1μM）通过快速灌注（2毫升/分钟），持续1分钟，然后再洗涤尼古丁出用HBS鸡尾酒。保持捕获时间推移图像之前，期间和烟碱施用后每2秒5分钟。 </li><li>量化前FM1-43荧光强度（F 染色 ）和后（F 脱色 ）在每个突触布顿烟碱应用。 </li><li>由前和尼古丁应用（ΔF= F 染色 -F 脱色 ）后定量FM1-43荧光强度的差计算沿vHipp轴突释放的FM1-43荧光总量。尼古丁下面的公式后分析的荧光强度降低分数：F 减少 （％）=ΔF/女染色 。 </li></ol> 4.钙成像<ol><li>与正常HBS快速- （7天在体外 5），然后加载与文化5微米荧光- 4 钙指标（冲洗文化调幅酯）和0.02％的Pluronic F-127在HBS（2ml）中进行30分钟，在37℃和5％的CO 2。 </li><li>洗出的Fluo-4溶液用HBS（3次/ 5分钟）。返回培养孵化器（37℃/ 5％CO 2）为至少30分钟。 </li><li>文化保持在相同的成像室，并在基于FM1 - 43水泡融合节所述相同条件下（见步骤3.1）。 </li><li>收集来自vHipp微片，用计划复消色差透镜物镜（60X水与1.4的NA，激励488纳米，发射530纳米）和CCD照相机的轴突突起的Fluo-4的荧光图像。 </li><li>收集基线的Fluo-4的荧光图像（每10秒，2分钟）作为预尼古丁控制，应用尼古丁（1μM）通过快速灌注（2毫升/分钟），持续1分钟，然后再洗涤尼古丁出用HBS鸡尾酒。保持捕获时间推移图像之前，期间和烟碱施用后，每10秒30分钟。 </li><li>保存原始FLUO-4的荧光图像，出口的所有帧作为一系列TIFF格式的图片进行进一步分析。 </li><li>收集荧光 - 4荧光沿轴突vHipp前和尼古丁申请后的整合力度。 </li><li>正常化集成荧光强度用下列方程：ΔF/ f 0的 =（FF 0）/ F 0，其中F 0是背景校正的预尼古丁集成荧光强度和F是在每个时间点沿vHipp轴突的积分荧光强度尼古丁后应用。分析并绘制标准化综合荧光强度。 </li></ol>

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作者什么都没有透露。

共培养制备所述重新投降体外腹侧海马伏电路。该制剂允许相对简单和可靠的空间和时间曲线通过该激活突触前烟碱受体引起增强谷氨酸传输5,21检查。 共培养物被定义为不同的特定细胞类型的在一个菜的生长，可以提供在生理条件的体外证明体内样功能。常规神经元的神经元共培养相继出台神经科学的研究为研究不同类型的细胞之间突触?...

电路的兴奋性胆碱能调制有助于认识的基本方面，并改变胆碱能调制的神经变性和神经精神障碍的特征，包括阿耳茨海默氏病，帕金森氏病，精神分裂症和成瘾1-4。建立突触传递在中枢神经系统的胆碱能便利化机制是通过直接激活烟碱受体定位在突触前部位。这些突触前受体的激活导致增加的细胞内Ca 2+（内[Ca 2+] i）的突触前端子-包括直接，由于某些胆碱受体亚型，并间接的相对高的钙传导，通过细胞内的信号级联5，由此增强神经递质释放。事实上，突触前烟碱受体的激活已与变化的各种各样的神经递质包括谷氨酸，GABA，乙酰胆碱，释放一个第二多巴胺6-10。尽管间接使用电生理学方法在各种突触这一过程进行了研究，光学记者的[Ca 2+] i和突触小泡循环允许更直接的和时间上精确测量的突触前的现象。 烟碱受体的突触前本地化已被证明具有令人信服的烟碱受体在电子显微镜（EM）水平11,12直接免疫金标记。几个其它技术也被用于间接寻址nAChR的定位，包括：检测在培养的神经元13,14的nAChRs的亚单位的荧光蛋白嵌合体的位置，电生理记录的nAChR电流在突触末端15,16，监测尼古丁引起的变化的[Ca 2+] 我由活细胞成像17，和神经递质释放的间接监控在由突触终端突触神经末梢用荧光指示剂活细胞成像的技术，包括由苯乙烯两亲调频染料（FM1-43和FM4-64）和/或突触-pHluorin与由特定的荧光神经递质记者，如CNiFERs为乙酰胆碱和iGluSnFr谷氨酸观察突触小泡的胞吐18-20。总体而言，目前的这些方法鉴定烟碱受体的突触前本地化是复杂的，并且需要特殊的系统和技术，让可靠的鉴定和生理监测突触前活动。 在这里，我们描述的协议和设备进行了腹海马（vHipp）的体外共培养体系-伏隔核（NACC）电路，可直接访问识别和分析前和突触后突触传递的组成部分。我们发现烟碱受体的突触前本地化和胆碱受体的活细胞成像介导的钙信号和神经递质的释放ALO的例子NG vHipp轴突。这里介绍的协议的一个自然（和直接的）扩展是由来自不同基因型的神经元前和突触后接触的制备方法。以这种方式，特定的基因产物，以调制的前和/或突触后的机制的贡献可以直接评估。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
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		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
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		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
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		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
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		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
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		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

所使用的制剂包括基因嵌合体外 vHipp-NACC电路的共培养物。从预测microslices vHipp发出，作为突触前神经轴突的输入，可与突触后的目标，从NACC分散的神经元突触联系。尼古丁引起的由vHipp支配神经元NACC持续（≥30分）促进谷氨酸传输轴突21和长期钙通过突触前α7nAChRs的*沿轴突vHipp信号5。 图1显示了直接从vHipp微切片的投影是谷氨酸（VGLUT1正）...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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8.9K 次观看.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.